Kit for luciferase assay

Abstract

Methods and compositions are provided for improved kinetics of light production from luciferase activity in beetle luciferase-luciferin reactions. Thus, the invention provides methods, compositions and test kits for assaying samples for the presence of a beetle luciferase or, using a beetle luciferase-luciferin reaction, the presence of ATP. The invention also provides the complex of the coenzyme, Coenzyme A, a beetle luciferase and oxyluciferin in its exited state in the luciferase-luciferin reaction and luciferyl-CoA, the thioester of Coenzyme A and luciferin (D-(-)-2-(6'-hydroxy -2'-benzothiazolyl)-Δ 2 -thiazoline-4-carboxylic acid).

I claim: 
     
       1. A test kit for assaying for the presence of a bettle luciferase in a sample thought to contain such a luciferase, said kit comprising a luciferase-luciferin reaction composition comprising a thiol reagent other than CoA, adenosine triphosphate, luciferin, and magnesium ion, said reaction composition being free of luciferase. 
     
     
       2. A test kit according to claim 1 for assaying for the presence of a bettle luciferase in a sample of cultured mammalian cells though to contain such a luciferase, said kit comprising additionally, and separately from the luciferase-luciferin reaction composition, a composition for lysing said cells, said lysing composition being an aqeous solution at a pH and ionic strength at which the luciferase is active in the luciferase-luciferin reaction and comprising a non-ionic detergent at a concentration between 0.1% and 10% (w/w), a proteinaceous luciferase-activity enhancer at a concentration between 0.1 mg/ml and 5 mg/ml, a thiol reagent other than CoA at a concentration between 1 mM and 10 mM, and glycerol or ethylene glycol at a concentration between 1% and 50% (v/v). 
     
     
       3. A test kit according to claim 1 wherein said lucifrase-luciferin reaction composition is an aqueous solution, which comprises CoA at between 0.1 mM and 1.0 mM, a thiol reagent other than CoA at between 5 mM and 100 mM, adenosine triphosphate at between 0.10 mM and 1.0 mM, luciferin at between 0.1 mM and 1.0 mM, and magnesium ion at between 2 mM and 15 mM. 
     
     
       4. A test kit according to claim 3 wherein the thiol reagent other than CoA is dithiothreitol, which is present in the solution at a concentration between 5 mM and 100 mM. 
     
     
       5. A test kit according to claim 3 for assaying for the presence of a beetle luciferase in a sample of cultured mammalian cells thought to contain such a luciferase, said kit comprising additionally, and separately from the luciferase-luciferin reaction composition, a composition for lysing said cells, said lysing composition being an aqeous solution at a pH and ionic strength at which the luciferase is active in the luciferase-luciferin reaction and comprising a non-ionic detergent, which is an octoxynol mixture with the average number of ethylenyloxy units per molecule between 9 and 10, at a concentration between 0.1% and 10 % (v/v), bovine serum albumin at a concentration between 0.1 mg/ml and 5 mg/ml, dithiothreitol at a concentration between 1 mM and 10 mM, and glycerol or ethylene glycol at a concentration between 1% and 50 % (v/v).